| Nano drug delivery systems have been proved effective in decreasing side effects of tumor chemotherapy. Since late20th century, rapid advances have been developed this technology. Nano-materials, as targeting drug carrier, have also been thrown an unprecedented attention on. Nano-drug formulations in cancer treatment and diagnosis are showing great promise. However, the shortcomings of traditional nano drug delivery systems were obvious, such as drug leakage, low drug loading capacity, poor targeting, et al.Poly(amidoamine)(PAMAM) is a kind of dendrimer with a great number of active groups on its surface and easy to be modified. There are many caves inside it which can embed drug. The molecular can set up an indeed nanoparticle (NP) with it self thus the drug leakage when nanoparticles of multimolecule collapsed could be avoided. As same as other nano drug delivery systems, some problems such as uptaking by reticuloendothelial system (RES), lack of stability in body and poor targeting were also found in PAMAM. It’s important to enhance its tumor targeting capbility.As to make up those shortcomings, we set up to design a new material. According to the pathological feature in tumor:higher vessel permeability, acid and highly expressed foliate receptor, we designed a new material. The new material was designed with PAMAM as the core to improve drug loading capacity as it possesses a large number of caves in the molecule. The end of PAMAM was connected to a pH-sensitive material, polymethacrylate (PMA), so that the drug would be released in the tumor pH environment but not in the normal tissue. The external end of polymethacrylate was connected to polyetheyloxygen (PEG) to avoid recognition by RES to achieve the purpose of long-cycling. As the same time, PEG can increase distribution in tumor through EPR effect. Folic acid (FA) was used as end group to achieve active targeting feature to tumor cells. The material was designed to enhance tumor targeting by the combination of pH-sensitivity, passitive targeting and active tumor targeting of FA.The synthesis began with the combination of FA and PEG. The FA-PEG was then combined with S-isobutyl-S’-(a,a’-dimethyl-a"-acetic acid)trithiocarbonate (BDAT) to be a polymer initiator of reversible addition fragmentation chain transfer (Raft) polymerization. Methyl methacrylate monomers were polymerized to the BDAT end of the initiator to be a two block polymer. Then, the BDAT end of polymer was aminolized to hydrosulfide.4-pentenoic acid (PA) was combined to the terminal amino group of PAMAM to get a double bond end. The double bond end of PAMAM-PA and the hydrosulfide of the two block polymer combined through addition. The linear polymer and the PAMAM core assembled to get the aimed polymer. The synthesis route was proved feasible and the structure of product was under control.The nano drug delivery system was designed according to the different pH between tumor and normal tissue. It should release drug rapidly in tumor (pH<6) and not release in normal tissue (pH-7.4). The critical pH point of polymethacrylate depends on the kind and ratio of monomers. So the copolymers of DMAEMA and2-droxyethylmethacrylate (HEMA), n-butylmethacrylate (BuMA), hexyl methacrylate (HMA),2-ethylhexyl methacrylate (EHMA) were screened by swell property in different pH. Then the copolymers were made into NPs loading paclitaxel (PTX) and their release character in different pH was researched. The final monomer kind and ratio of monomers in pH-sensitive block was decided as DMAEMA-BuMA50:50.This article also examined the effect of PEG on the pH sensitivity. When connected with water-soluble PEG, the release rate of PTX decreased. The pH sensitivity changed not so obviously in the scope of this study. Therefore we used PEG (5000) at last.Reversible release experiment was practiced between the dendrite material and the linear mid-product. The results showed that the NP prepared of the dendrite material possessed faster response speed to the changed pH and better reversibility than that of the linear mid-product, thus the design was verified reasonable. The final material was FA-PEG-PMA-PAMAM with the molecular of PEG5000and the pH-sensitive block of DMAEMA-BuMA50:50.The preparation method of PTX loading NP was initially identified as the emulsion-solvent evaporation method. The parameters were determined by orthogonal experiments. The encapsuled ratio (ER) was more than95%and the drug loading (DL) was more than4.6%. The particle size in water was about100nm with a narrow distribution. The particle size increased as the pH changed from7.4to5.0. Anti-macrophage phagocytosis test was used to evaluate the long-circulating character of the drug delivery system. The results showed that the anti-phagocytic ability of the NP of FA-PEG-PMA-PAMAM was significant stronger than that of PAMAM. The introduction of PEG decreased the phagocytosis, phagocytosis of linear material was less than dendrite materials, of modified PAMAM less than of PAMAM itself. In addition, FA showed no significant effect against anti-phagocytosis. The long-circulating feature has been proved on cell level.FA sensitive tumor cell KB was used to evaluate the tumor targeting feature of the NPs. The ratio of uptake ratio in non-folic acid medium and normal medium suggested that the trend of FA-PEG-PMA-FA-PEG-PMA-PAMAM> PEG-PMA-PEG-PMA-PAMAM> PMA-PMA-PAMAM> PAMAMA. There was no significant difference between dendrite and linear materials of the same composition.L929cell was used to evaluate the cell compatibility of FA-PEG-PMA-PAMAM. The safe dose was below625μg/ml.KB cell was used to evaluate the cell toxicity of NP. The PTX-NP of FA-PEG-PMA-PAMAM showed stronger toxicity in non-FA DMEM. The toxicy dose is10μg/ml of PTX, equal to200μg/ml of dendrimer when made into nanoparticle.The pharmacokinetics of PTX loading NP in rats and the distribution in KB-bearing nude mice was determined by LC-MS/MS method to verify the long circulating and tumor targeting capability. The experiments were practiced with PTX injection, PTX loaded FA-PEG-PAMAM and PTX loaded FA-PEG-PMA-PAMAM.In the compartment model parameters, distribution and elimination half-life ti/2α, t1/2β and MRT (0-t), MRT (O-∞)of NP were significantly higher than the free PTX., reflecting the longer retention time than free PTX. AUC (0-t) and AUC (0-∞) of FA-PEG-PMA-PAMAM NP were significantly higher than those of free PTX and FA-PEG-PAMAM NP, suggesting the pH sensitive release of FA-PEG-PMA-PAMAM NP prolonged the retention time of PTX.Result of tissue distribution experiment proved tumor targeting feature of FA-PEG-PMA-PAMAM NP. NPs distributed more slowly after injection than the free-PTX. The PTX concentrations of NPs were higher than free PTX in liver, lung and kidney. There was no difference in spleen. The PTX concentrations of NPs were higher than free PTX in tumor. The targeting efficiency of NP was more than 3compared with the free-PTX and about1.8compared with FA-PEG-PAMAM NP, which means the targeting capacity of FA-PEG-PMA-PAMAM is higher than that of FA-PEG-PAMAM. The reason maybe is that the pH sensitive release of FA-PEG-PMA-PAMAM NP could protect the PTX, thus the opportunity of return to the circle and arrive at the tumor increases.The results of pharmacokinetics and distribution experiments proved the long circling and tumor targeting capacity of FA-PEG-PMA-PAMAM.The pharmacodynamic study was practiced in KB tumor subcutaneously imbed nude mice. Through the monitoring of tumor volume, tumor weight and the organ ratio statistics, the results were displayed. The results showed significant inhibition of KB cells in nude mouse tumor growth after3to15times injection of the PTX loading FA-PEG-PMA-PAMAM NP with the dosage of1mg/kg. The inhibition was significantly higher than free PTX. The inhibition ratio was about70%. After administration of15times, there was no obvious injury in heart, liver, spleen, lung and kidney in biopsy.The intravenous injection LD50of mice was35.23mg/kg. The NP showed no obvious irritation in rabbit ear vein after5time injection.The study suggests that the project design and synthesis of the carrier material was successful. The material possesses multi-level tumor targeting character of natural targeting, physical targeting, long circulating propertie and active targeting. It may be a new idea for therapy of FA sensitive tumors. |
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